Template:Waterloo/notes

var notes = {"Jun28": ["Digest CFP_Gal1 PCR and Hsp104 Plasmid with BamHI and XhoI 1000ng, 30min digest", "Column purify both fragments with PCR Clean up kit", "Ligate fragments together using 4:1 ratio", "Write up Experimental Flow of oePCR group and approve it with Patrick", "Re organize Sq A workbox, following the Group Working Box tab ", "Autoclave a 500mL baffled flask holding 250mL LB", "1 Inoculate 250mL LB with 5mL culture of DH5a, no antibiotics, grow till OD 0 4 0 5", "2 Pellet using 3K RPM, 10min, 4C; decant; resuspend in 125mL 0 1M MgCl2, inc at 4C for 30min", "3 Pellet using 3K RPM, 10min, 4C; decant; resuspend in 125mL 0 1M CaCl2, inc at 4C in ice + o/n", "Inoculate DH5a into 5mL LB w/ set of antibiotics to check for contamination", "Autoclave more 1 5mL tubes, 2 0mL tubes, and the inoculation sticks in the flowhood "], "Jun29": ["1 Inoculate 250mL LB with 5mL culture of DH5a, no antibiotics, grow till OD 0.4-0.5", "2 Pellet using 3K RPM, 10min, 4C; decant; resuspend in 125mL 0 1M MgCl2, inc at 4C for 30min", "3 Pellet using 3K RPM, 10min, 4C; decant; resuspend in 125mL 0 1M CaCl2, inc at 4C in ice + o/n", "Inoculate the DH5a from the LB-only culture into 5mL LB w/ 2.5uL Km", "Resuspend ADH1:GFP synthesized fragment.", "PCR amplify the ADH1:GFP fragment using Primers 5 & 6", "Diagnostic gel on the PCR product, 1% agarose gel run at 80V ", "Use PCR Clean up kit for the PCR product +Nanodrop", "Put the autoclaved tubes and inoculation sticks in the dry oven for the day.", "Troubleshoot the oePCR on SnapGene", "Transform Hsp104-CFP Ligation into DH5alpha. Use Hsp104 plasmid as a +Ve control (antibiotic is Amp)", "Make 50mL Adenine solution (5mg/mL)", "Make 50mL Tryptophan solution (10mg/mL)", "Make 50mL Uracil Solution (2mg/mL)", "Make 50mL Histidine solution (10mg/mL)", "Make 50mL Leucine Solution (10mg/mL)", "Bring TEL, PEG-TEL, and other transformation reagents to iGEM lab ", "Prepare plates for replica plating experiment tomorrow ", "Make 1L of WO media and autoclave ", "Wash glassware in the sink"], "Jun20": ["Organize SnapGene files ", "Rearrange & order 4 remaining IDT sequences ", "Dilute primers in TE to concentration of 100uM ", "Get liquid nitrogen", "Make long term storage stock of plasmid cultures Hsp104, Sup35, pXP", "Do miniprep of Hsp104/Sup35 plasmid", "Done Biology Technical document", "Clean glassware"], "Jun21": ["Rearrange & order 4 remaining IDT sequences ", "PCR of Hsp104 plasmid to remove first fragment for OE PCR use temp gradient", "Make 0 8% agarose gel, 11 wells ", "Run gel to check for correct band size in PCR, run at 100V"], "Jun22": ["Inoculate pXP218 in 5mL LB w/ 2 5uL Amp x2", "Complete the LD Leads Planner"], "Jun23": ["Diagnostic d digest of pXP218 BamHI & SalI & Hsp104 BamHI & BglII ", "Diagnostic 1% ag gel run w/ conditions for clear bands, ie nice and slow for the d digest ", "Make YPD Plates 20 plates using the steamer and store in the fridge", "Re streak PSI+ strain on a YPD plate it looks contaminated", "Make Frozen stocks of PSI , PSI+, and CFP yeast strains", "Resuspend synthsized fragment CFP_Gal1 in appropriate buffer"], "Jun24": ["Resuspend synthesized fragment from IDT in appropriate buffer ", "PCR of CFP_Gal1 promoter using primers 7&8 use temperature gradient ", "Make small 1% agarose gel ", "Run PCR product on a gel to check for correct band size ", "Do PCR cleanup on CFP_Gal1 PCR Product an nanodrop", "Pick up enzymes from Terence ", "PCR with primers 3&4 to amplify CFP for o/e PCR ", "PCR Cleanup of Hsp104 PCR to prepare for o/e PCR", "Make small 0 8% agarose gel for running CFP ", "Run PCR product on 0 8% agarose gel to check for correct CFP band size"], "Jun27": ["Digest CFP_Gal1 PCR and Hsp104 Plasmid with BamHI and XhoI 1000ng, 30min digest", "Column purify both fragments ", "Ligate fragments together using 4:1 or 5:1 ratio this will be updated", "Update the Status of the Tasks from June 23rd ", "Fill ten 10uL and 200uL tip boxes, autoclave, and put in the drying oven O/N", "PCR Clean up of CFP fragment pooled together for Sq A/Grp 2, for oePCR +Nanodrop"], "Jul28": ["Finish gel extraction, remove DNA from gel fragment", "Ligate together the Hsp104 and Gal1:CFP o/n.", "Blunt Hsp104 fragment", "Column purify blunt Hsp104 and nanodrop", "Pick colonies from the \"product\" plate of pSB and pXP to be grown overnight in LB with antibiotic", "Replica plate Transformants and viability plates(+URA, +ADE - URA, -ADE - URA)"], "Jul29": ["Transform E.coli with Hsp104_CFP-Gal1 batch 2", "Plate Hsp transformed E. coli on Amp plates", "Resuspend CFP_Stop1 and CFP_Stopcodon2", "PCR CFP stopcodons 1 and 2", "Run PCR products on a 1.2% gel and run at 80V ", "Digest 2000ng Hsp104 with ApaI and BamHI at 16C", "Gel extract large band from Hsp104, run on 1% gel at 80V, image gel AFTER digest is removed", "Blunt Hsp104 with Taq polymerase and hot start buffer at 37C for 2 hours", "Column purify blunt Hsp104", "Digest CFP stopcodon 1 and 2 with just BamHI", "Ligate blunted Hsp104 to CFP stopcodon 1 and 2 (seperately)", "Miniprep pSB and pXP cultures in the incubator (from transformation yesterday) and nanodrop", "Digest pSB plasmids (minipreped earlier today) with...", "Digest pXP plasmids (minipreped earlier today) with...", "Run pSB and pXP diagnostic digests on 1% gel at 80V", "Replica plate Transformants and viability plates(+URA, +ADE - URA, -ADE - URA)", "Troubleshoot PCR for Cup1 and Gal1", "Fix Holtin"], "Jul22": ["Finish new restriction digest protocol for Hsp104 with BamHI, ", "Run digest and polymerization of Hsp104", "Do diagnostic digest of 200ng of Hsp104 with ApaI in CutSmart", "PCR Clean-up of the ADH1 pcr products that worked, then nanodrop.", "Clean dishes (mugs, cups) in the sink next door", "Remove the test tubes from the oven", "Viability Assay on yeast from last 3 days "], "Jul23": ["Count the number of colonies on the plates from the Viability Assay", "Digest Hsp104 w/ ApaI at 16C for 60min", "Run a 1% diagnostic gel on the digested Hsp104."], "Jul20": ["Digest pSB1C3 (200ng) w/ just XhoI for 30min at 37C", "Run a diagnostic 1% gel of the pSB1C3 digest and look for band lengths: 900bp and 1.2kb", "Check if XhoI site on Hsp104 is methylated try to find a/some resources on how to check", "PCR ADH1 w/ Pat's recipe/condtions, this time he does it with the Q5MM and GoTaqMM to compare.", "Run a diagnostic 1% gel of the PCR product, looking for a band at 1.7kb, 70V", "PCR Clean-up of the ADH1 pcr product, then nanodrop.", "Take sample of PSI- and Sup35 at 11:00 PM", "Take sample of PSI- and Sup35 at 2:00 PM", "Take sample of PSI- and Sup35 at 5:00 PM", "Finish cleaning the test tubes, and autoclave.", "Throw-out the biohazardous waste, making sure to tie the yellow bag before you leave the lab."], "Jul21": ["Image diagnostic 1% gel of the pSB1C3 and Hsp104 digest and see what you get. ", "PCR amplify ADH1 using Patricks conditions from yesterday", "PCR Clean-up of the ADH1 pcr products that worked, then nanodrop.", "Take 1ml yeast sample at 11 AM, one from each flask, spin down and resuspend in 1mL DI", "Take 1ml yeast sample at 2 PM, as above", "Take 1ml yeast sample at 5 PM, as above", "Clean the glassware in the sink, rinse with DI.", "Remove the test tubes from the autoclave and place in oven o/n"], "Jul26": ["Gel extract the larger Hsp104 fragment on a 1% gel run at 80V, then nanodrop.", "Add Taq DNA polymerase and dNTPs to the gel extracted volume of Hsp104.", "Column purify DNApol-treated Hsp104 w/ spin columns, then nanodrop.", "Ligate together the Hsp104 and Gal1:CFP o/n.", "Column purify ADH1:GFP and pSB1C3 w/ spin columns, then nanodrop.", "Ligate the ADH1:GFP and pSB1C3 together.", "Column purify ADH1:GFP and pXP218 w/ spin columns, then nanodrop.", "Ligate the ADH1:GFP and pXP218 together.", "Run a PCR of Gal1:GFP on a temperature gradient", "Check that we're stocked on Cm and Amp plates. If not, make 20 of the ones we're low on.", "Viability Assay Analysis - just count plates and record numbers in book", "Viability Assay Analysis: make excel file to hold this round of data", "Replica plate Transformants and viability plates(+URA, +ADE - URA, -ADE - URA)", "Make new primers 5&6", "Resuspend Cup1_promoter in 100 uL TE buffer"], "Jul27": ["Digest 2000ng Hsp104 with ApaI, and BamHI in cutsmart at 16C for 1.5 hours, do a control with just BamHI", "Gel extract the larger Hsp104 fragment on a 1% gel run at 80V", "Add Taq DNA polymerase and dNTPs to the gel extracted volume of Hsp104.", "Column purify DNApol-treated Hsp104 w/ spin columns, then nanodrop.", "Ligate together the Hsp104 and Gal1:CFP o/n.", "Transform and plate with the ligated pXP218 and pSB1C3 from yesterday", "PCR amplify Cup1 and Gal1_Promoter using primers 5&6 at 63C", "Run 1% diagest gel at 80V of Cup1 and Gal1", "Replica plate Transformants and viability plates(+URA, +ADE - URA, -ADE - URA)"], "Jul25": ["Digest 2000 ng of Hsp104 with BamHI at 37C for 40 minutes in cutsmart buffer", "Digest the same Hsp104 from above with ApaI at 16C for 1 hour", "Digest Gal1:CFP with only BamHI, preferably 500ng if possible.", "Gel extract the larger Hsp104 fragment on a 1% gel run at 80V, then nanodrop.", "Column purify Gal1:CFP w/ spin columns, then nanodrop.", "Add T4 DNA polymerase and dNTPs to the gel extracted volume of Hsp104.", "Column purify DNApol-treated Hsp104 w/ spin columns, then nanodrop.", "Ligate together the Hsp104 and Gal1:CFP o/n.", "Resuspend the IDT Gal1:GFP fragment in TE.", "PCR amplify the Gal1:GFP, and ADH1:GFP fragment with primers 5 and 6 with a Ta of 63C.", "Diagnostic (1%) gel of the Gal1:GFP amplified fragment (1uL of the PCR rxn volume only).", "Digest ADH1:GFP and pSB1C3 w/ E and P (~200ng and 2000ng, respectively).", "Column purify ADH1:GFP and pSB1C3 w/ spin columns, then nanodrop.", "Ligate the ADH1:GFP and pSB1C3 together.", "Digest ADH1:GFP and pXP218 w/ SphI and SalI (~200ng and 2000ng, respectively).", "Column purify ADH1:GFP and pXP218 w/ spin columns, then nanodrop.", "Ligate the ADH1:GFP and pXP218 together.", "Check that we're stocked on Cm and Amp plates. If not, make 20 of the ones we're low on.", "Viability Assay Analysis", "Re streak transformants (2 from each plate) onto YPD plates ", "Organize working boxes"], "Aug31": ["Transform the CFP NSC/SC1/SC2-Hsp104 Batch 2", "Transform the Cup1/Gal1-pXP Batch 3", "Transform the ADH1/Gal1-pSB Batch 2", "Analyze transformation products and determine which ones can have cPCR.", "Transform the sgRNA-Main & RFP-CTRL Batch 1", "Attempt 1: cPCR CFP NSC/SC1/SC2-Hsp104 Batch 1 (72 colonies)", "cPCR Cup1/Gal1-pXP Batch 1 (~8 colonies)", "cPCR Cup1/Gal1-pXP Batch 2 (~8 colonies)", "cPCR ADH1/Gal1-pSB Batch 1 (~8 colonies)", "Make ~30 Amp plates", "Make 20 Cm plates", "Digest 2000ng of pXP218 with SphI and SalI X2. Digest for 2h at 37C ", "Gel extract 6.2kB fragment after running on a gel with guanosine. There should be only 1 band "], "Aug30": [], "Jul17": ["Miniprep the 3 Hsp104 tubes, then nanodrop.", "PCR amplify Gal1-CFP w/ previous successful recipe/conditions w/ 1 -ve CTRL.", "Diagnostic digest 200ng of Hsp104 w/ BamHI and XhoI separately; inc. 37C for 30min.", "Run a 1% gel of the digested Hsp104 (preferably 1uL of Sample with 4uL of Water and 1uL of LD.", "Re-organize and maintain the workboxes in the -20C freezer.", "Wash glassware in the sink, and clean-up the lab benches."], "Jul16": ["PCR Clean-up of the Gal1_CFP in the pink tray of 4C", "Inoculate Hsp104 into 3 tubes of 5mL LB w/ appropriate antibiotics.", "Digest Gal-CFP and Hsp104 vector (>1000ng each) with BamHI and XhoI.", "Gel extract both fragments in a 1% gel and nanodrop.", "Ligate the Gal-CFP w/ Hsp104 using two negative controls. Incubate o/n at RT.", "Re-organize and maintain the workboxes in the -20C freezer.", "Wash glassware in the sink, and clean-up the lab benches.", "Autoclave 1.5 ml MC tubes, innoculation sticks, pippette tips, Mili-Q, anything else that needs to be topped up"], "Jul15": ["Make 50 YPD Plates for plating cultures today - YPD is made just put it in the steamer.", "Take aliquots every 1.5h starting 11:30 of the cultures until 6h at 5:30pm.", "Do Viability assays on yesterdays and todays cultures on YPD.", "PCR amplify Gal1_CFP, 2 sets and 1 -ve CTRL", "Digest Gal-CFP and Hsp104 vector (>1000ng each) with BamHI and XhoI.", "Gel extract both fragments in a 1.2% gel, using the 20:1000 LD, and nanodrop.", "Ligate the Gal-CFP w/ Hsp104 using two negative controls. Incubate o/n at RT.", "Dilute PAT's LD 20:1000uL, followed by organization of the \"Gel Stuff\" box in the -20C freezer.", "Re-organize and maintain the workboxes in the -20C freezer.", "Call NEB and troubleshoot the PCR smearing from their clean-up kit.", "Add agar to the LB bottles Emma made, then autoclave.", "Make 2L of LB Agar and autoclave today"], "Jul14": ["PCR amplify Gal1_CFP, 5 sets and 1 -ve CTRL", "PCR Clean-Up of Gal1_CFP, followed by nanodrop.", "Digest Gal-CFP and Hsp104 vector (>1000ng each) with BamHI and XhoI.", "Gel extract both fragments in a 1.2% gel, using the 20:1000 LD, and nanodrop.", "Ligate the Gal-CFP w/ Hsp104 using two negative controls. Incubate o/n at RT.", "Dilute PAT's LD 20:1000uL, followed by organization of the \"Gel Stuff\" box in the -20C freezer.", "Diagnostic 1% gel of the ADH1 (5uL w/ 1uL LD) straight from the synthesis IDT tube again.", "Make 2L worth of LB in 200mL containers. ", "Consult Cheng and John about the ADH1::GFP amplification problems.", "Re-organize and maintain the workboxes in the -20C freezer.", "Remove the recycling and garbage from the office. ", "Remove the transformation plates from the 37C inc. and check for colonies."], "Jul13": ["Gel extract both fragments in a 1.2% gel, using the 20:1000 LD, and nanodrop.", "Ligate the Gal-CFP w/ Hsp104 using two negative controls. Incubate o/n at RT.", "Diagnostic gel of the ADH1 (5uL w/ 1uL LD) straight from the synthesis IDT tube again.", "PCR amplify the ADH1:GFP fragment using Primers 5 & 6", "Make 20 Km plates, some for SHAD and some for us to keep. ", "Dilute PAT's LD 20:1000uL, followed by organization of the \"Gel Stuff\" box in the -20C freezer.", "PCR amplify Gal1_CFP, 5 sets and 1 negative control, use 0.5uL DNA template, 70C annealing, 45s elongation", "Inoculate 5X of Sup35 transfomed yeast in 5mL, 5X Psi- in 5mL for tomorrow experiment - put in 30C with shaking"], "Jul12": ["Gel extract both fragments in a 1.2% gel, using the 20:1000 LD, and nanodrop.", "Ligate the Gal-CFP w/ Hsp104 using two negative controls. Incubate o/n at RT.", "Make NEW primer stocks for 5 & 6 under the flame with 0% contamination.", "Diagnostic gel of the ADH1 (5uL w/ 1uL LD) straight from the synthesis IDT tube again.", "PCR amplify the ADH1:GFP fragment using Primers 5 & 6", "Make 30 Cm plates, some for SHAD and some for us to keep. ", "Dilute PAT's LD 20:1000uL, followed by organization of the \"Gel Stuff\" box in the -20C freezer.", "Clean-up the top of the table sitting above the microwave (throw-out the random bags and the like)", "Fill up the large jug with MilliQ from Terrence", "Replace the white lab bench paper at the chemicals section with new bench paper. ", "Clean glassware in the sink (it's not even Wednesday yet and there's so much LOL)."], "Jul11": ["Nanodrop the nine miniprepped samples and record in the lab book.", "Run a diagnostic digest with EagI and XhoI.", "Run a 1% gel on the digested samples, 80V for ~45mins, image, and put in lab book. ", "Make frozen stock of the samples which have the correct insert size. ", "Inoculate new samples of the successful transofmration into LB w/ appropriate antibiotics.", "PCR amplify the ADH1:GFP fragment using Primers 5 & 6", "Diagnostic gel on the PCR product, 1% agarose gel run at 80V ", "Use PCR Clean up kit for the PCR product +Nanodrop", "Diagnostic gel of ADH1 straight from the synthesis tube.", "Clean-up the top of the table sitting above the microwave (throw-out the random bags and the like)", "Fill up the large jug with MilliQ from Terrence", "Replace the white lab bench paper at the chemicals section with new bench paper. ", "Miniprep 284, then nanodrop.", "Miniprep 322, then nanodrop.", "Miniprep 326, then nanodrop.", "Digest 284 w/ XbaI and PstI for 60min @ 37C", "Digest 326 w/ SpeI and PstI for 60min @ 37C", "Gel extract 1kb fragment from 284, and the single band (~2kb) from 326, then nanodrop.", "Make 1L YPD Agar, autoclave, then pour plates and store them at 4C ", "Make 1L YPD broth, aliquot 100mL into 10 250mL Baffled flasks, then autoclave ", "Replica plate the streak plates with +URA/-URA to test for the plasmid. ", "Inoculate 5 tubes each of the PSI- strain and the PSI- with sup35 for experiment tomorrow", "Depending if Hsp104-CFP looks good, inoculate 5 tubes of PSI- to transform into yeast tomorrow"], "Jul10": ["Inoculate 284, 322, and 326 into appropriate antibiotics (x5 each)"], "Jul19": ["Run 1.2% gel of digested Hsp104 at or after 2:00 PM", "PCR of ADH1 at 63C, using Patrick's conditions found in lab book.", "Run 1.2% gel of ADH1 pcr product at 80V", "PCR Clean-up of the ADH1 pcr product, then nanodrop.", "Miniprep the pSB1C3 and pXP218 from the 37C incubator (x6 tubes in total), then nanodrop.", "Inococulate 100ml YPD with yeast PSI- and Sup35, normalize to OD of 1.0, add 60uL of CuSO4", "Make 2L of YPD and agar and autoclave", "Make 80 YPD plates", "Take 1mL sample from Sup35 and PSI- at 5:00PM, spin down, resuspend in 1 mL autoclaved mili-Q", "Clean test tubes", "Throw-out the biohazardous waste, making sure to tie the yellow bag before you leave the lab."], "Jul18": ["Troubleshoot the absence of XhoI cutting in the Hsp104 plasmid. ", "Diagnostic digest to check wtf is wrong with XhoI.", "Run a 1% diagnostic gel of the Gal1-CFP PCR from yesterday.", "PCR Clean-up of the Gal1-CFP from yesterday, then nanodrop.", "Re-organize and maintain the workboxes in the -20C freezer.", "Wash glassware in the sink, and clean-up the lab benches.", "PCR amplify ADH1:GFP fusion w/ Primer 5 & 6", "Diagnostic gel of ADH1:GFP amplifcation. ", "Count plates and do viability analysis", "PCR Clean-up of the successfully amplified ADH1 fragment, then nanodrop.", "Inoculate strain 111 into Cm, and pXP218 into Amp (x3 tubes each).", "Make excel file to fill out results of plate count", "Make YPD Agar, autoclave", "Make 80 YPD", "Inoculate PSI- and SUP35, 6 tubes each"], "Aug9": ["PCR CFP-Gal1, and CFP stopcodons 1 and 2", "Run PCR products on a 1.2% gel and run at 80V ", "Organize freezer", "PCR amplify Cup1 using primers 5&6", "PCR amplify Gal1 using primers 5&6", "Troubleshoot PCR for ADH1, Cup1, and Gal1", "Miniprep and nanodrop pSB-Gal1 and pSB-Cup1 from 5ml cultures in incubator", "Determine which enzymes to use for a diagnostic digest of pSB-Gal1 and pSB-Cup1", "Digest 200ng of pSB-Gal1 and pSB-Cup1 using previously determined enzymes", "Run diagnostic digest of pSB-Gal1 and pSB-Cup1 on 1% agarose gel, 1ul sample, 4 water, 2 dye. Use gel red", "PCR amplfy Gal1 and Cup1 using primers 17&18, only PCR a little bit, 10 ul max", "Run PCR products on a 1% gel at 80V", "PCR amplify gal1 and cup1 from previous step using primers 5&6", "Run PCR products on a 1% gel at 80V"], "Aug8": ["PCR CFP-Gal1, and CFP stopcodons 1 and 2", "Run PCR products on a 1.2% gel and run at 80V ", "Organize freezer", "PCR amplify Cup1 using primers 5&6", "PCR amplify Gal1 using primers 5&6", "Troubleshoot PCR for ADH1, Cup1, and Gal1", "Pick colonies from the pSB-Cup1 and pSB-Gal1 plate, 5 from each, innoculate 5ml broth with Cm", "Examin gels for ADH1 products (in pXP and pSB)", "Wash dishes", "Digest pXP-ADH1 with Pst1", "Run digested pXP-ADH1 on 1% gel"], "Aug3": ["Fix Zoltin", "PCR CFP stopcodons 1 and 2", "Run PCR products on a 1.2% gel and run at 80V ", "Organize freezer", "PCR amplify Cup1 using primers 5&6", "PCR amplify Gal1 using primers 5&6", "Troubleshoot PCR for Cup1 and Gal1", "Make 1:10 dilutions of 2014 primers 11&12", "PCR amplify Gal1 using primers 17&18 (from 2014)", "PCR amplify Cup1 using primers 17&18 (from 2014)", "Run 1% diagnosic gel of PCR products", "PCR clean up sucessful PCR products", "Digest Gal1, and Cup1 with EcoRI and PstI", "Digest 2000ng pSB with EcoRI and PstI", "Run digested pSB on a 1% gel, cut out and save top band", "Ligate pSB and Gal1 o/n", "Ligate pSB and Cup1 o/n", "Get 400ul gel red from John", "Ordered new primers for CFP stop codons "], "Aug2": ["Fix Zoltin", "PCR CFP stopcodons 1 and 2", "Run PCR products on a 1.2% gel and run at 80V ", "Digest 2000ng Hsp104 with ApaI and BamHI at 16C", "Gel extract large band from Hsp104, run on 1% gel at 80V, image gel AFTER digest is removed", "Digest pXP plasmids with PstI", "Run pXP diagnostic digests on 1% gel at 80V", "Troubleshoot PCR for Cup1 and Gal1"], "Aug5": ["Fix Zoltin", "PCR CFP stopcodons 1 and 2", "Run PCR products on a 1.2% gel and run at 80V ", "Organize freezer", "PCR amplify Cup1 using primers 5&6", "PCR amplify Gal1 using primers 5&6", "Troubleshoot PCR for ADH1, Cup1, and Gal1", "Transform and plate pSB-Gal1, incubate in 37 C overnight", "Transform and plate pSB-Cup1, incubate in 37C overnight", "Wash dishes", "Digest pSB1C3_ADH1 to check for insert", "Miniprep 4mL of 111 and 347 and make a frozen stock with the other 1mL - tell Alicia when done"], "Aug4": ["Fix Zoltin", "PCR CFP stopcodons 1 and 2", "Run PCR products on a 1.2% gel and run at 80V ", "Organize freezer", "PCR amplify Cup1 using primers 5&6", "PCR amplify Gal1 using primers 5&6", "Troubleshoot PCR for Cup1 and Gal1", "Finish gel extraction of digested pSB from section cut out of gel yesterday", "PCR clean up sucessful PCR products for Gal1 and Cup1", "Digest Gal1 with EcoRI and PstI", "Digest Cup1 with EcoRI and PstI", "Gel extract digested Cup1 and Gal1", "Ligate pSB and Gal1 o/n", "Ligate pSB and Cup1 o/n", "Autoclave tips", "Wash dishes", "Inoculate strain 111 (2.5uL Cm) and 347 (2.5uL Km), 1x each."], "Aug22": ["Miniprep 3mL of G1 and G2 to send for sequencing. Elute in STERILE WATER and wash 4X instead of 2X", "Make frozen stocks of G1 and G2 (CFP-SC2) and update the strain list ", "Prepare G1 and G2 samples for sequencing (refer to sequencing sheet)", "Send order for sequencing before noon ", "Design/Order new primers for Gene Retention, ADH1, CFP Biobricks, and pSB1C3 sequencing primers ", "Get paper towels from Chem Stores ", "Email Stacy Lavery about specifications of the fluorimeter for CFP testing ", "Autoclave tubes, wooden sticks in the morning. Use setting 1", "Make 2L of WO media, autoclave, then pour plates ", "Restreak all old yeast strains in the fridge onto new YPD plates. Label with initials and date.", "Ligate the backbone with CFP_Gal1, CFP-SC1 and CFP-SC2. Leave 2-4h at RT. (Row 4)", "Ligate the backbone with CFP_Gal1, CFP-SC1 and CFP-SC2. Leave 2-4h at RT. (Row 5)", "Ligate pSB1C3 with the three inserts. Leave 2-4h at RT.", "Ligate pXP218 with Cup1 and Gal1. Leave 2-4h at RT. ", "Transform CFP_NSC, CFP_SC1 and CFP_SC2 into DH5alpha. Antibiotic is Amp (Row 4)", "Transform CFP_NSC, CFP_SC1 and CFP_SC2 into DH5alpha. Antibiotic is Amp (Row 5)", "Make 20 Amp plates by steaming 2 LB Agar bottles", "Transform Gal1, Cup1, and Adh1 (pSB1C3) into DH5alpha. Antibiotic is Cm", "Transform Gal1, Cup1 (pXP218) into DH5alpha. Antibiotic is Amp ", "Make 20 Cm plates by steaming 2 LB Agar bottles", "PCR of Cup1 and Gal1 with primers 5&6 (50uL reation volumes, Patricks conditions)", "Run the products on a 1.2% gel to see if PCR worked ", "PCR Cleanup of the products and store at -20C ", "Make new amp and test by streaking 2 different cell types on it (pSB1C3 and Hsp104 plasmid) ", "PCR of Cup1/Gal1/Adh1 with primers 17/18 for pSB1C3", "Run products on a 1% gel to see if PCR worked ", "PCR Cleanup of the products and store at -20C - we will digest tomorrow ", "Inoculate 5X tubes of pXP218. Do a -ve control. Antibiotic is Amp "], "Aug23": ["cPCR on pSB1C3_Gal1 and pSB1C3_Adh1. Total 6 plates. Primers 19&20", "Run products on a 1% gel at 110V ", "Analysis on gel and determine if we need to send samples for sequencing ", "Check if Amp antibiotic is good by doing analysis on streak plates from yesterday ", "Make 10 Amp plates by using last LB agar bottle ", "Make 2L of LB Agar, put into 200mL bottles, autoclave (10 bottles total) ", "Make 1L of YPGal, 100mL in 5 baffled flasks and then 500mL into bottles ", "Grow 1L of DH5a to OD 0.4/0.5 ", "Autoclave 500mL 0.1M CaCl2 and GSA bottles. ", "Pellet the DH5a and resuspend in 500mL 0.1 MgCl2, then inc. at 4C for >1hr", "Pellet the DH5a and resuspend in 500mL 0.1 CaCl2, then inc. at 4C o/n.", "Inoculate Strain 284 into 5mL LB w/ 2.5uL Cm; 5 tubes.", "PCR of CFP_NSC, CFP_SC1, CFP_SC2. 50uL reaction volumes. Do a -ve control ", "Run products on a 1.2% gel to see if PCR worked - run at 80-100V ", "PCR cleanup on the products, nanodrop ", "Digest 1000ng of CFP_NSC, CFP_SC1, CFP_SC2 with ApaI and BamHI for 35 min at 37C ", "Column purification of CFP_NSC, CFP_SC1, CFP_SC2 and nanodrop ", "Digest 2000ng of Hsp104 plasmid X3 with ApaI and BamHI for 2h at 37C ", "Gel Extract 8.8kB fragment after running on a gel with guanosine. Other fragment should be 700bp", "Miniprep pXP 218 ", "Digest 2000ng of pXP218 with SphI and SalI X2. Digest for 2h at 37C ", "Gel extract 6.2kB fragment after running on a gel with guanosine. There should be only 1 band ", "Digest 1000ng of Cup1 and Gal1 (5&6 primers) with SphI and SalI for 35min at 37C", "Column purification of Cup1 and Gal1 then nanodrop ", "Digest 2000ng of pSB1C3 with EcoRI and PstI, incubate at 37C for 35min ", "Gel extract 2kB fragment after running on a gel with guanosine. Other fragment will be 1kB ", "Digest Adh1 and Gal1 (17&18 primers) with EcoRI and PstI. Incubate at 37C for 35 min", "Column purification of Adh1 and Gal1 then nanodrop ", "Ligate Adh1 and Gal1 into pSB1C3, leave at RT minimum 2h, 0.3uL ligase/rxn. Do 2:1 ratio insert:vector ", "Streak W303 psi- patch plates onto YPD for single colonies "], "Aug21": ["Design new construct plans for biobricking CFP-Gal1 and for Gene Retention ", "Inoculate G1 and G2 from the CFP_SC2 PCR plates for miniprepping and sequencing tomorrow ", "Make a sequencing spreadsheet and prepare the order for tomorrow (except for miniprepping) "], "Aug26": ["Colony PCR on CFP_NSC/SC1 (Vincent's Transformants from Aug 25). Use primers 13&14", "Run products on a 1% gel to check if the PCR worked ", "Analysis on cPCR results to see if we should send for sequencing ", "Colony PCR on CFP_NSC/SC2 (Sam's Transformants from Aug 25). Use primers 13&14", "Run products on a 1% gel to check if the PCR worked ", "Analysis on cPCR results to see if we should send for sequencing ", "Colony PCR on pXP218 Cup1 (Andres transformants from Aug 25). Use primers 15&16", "Run products on a 1% gel to check if the PCR worked ", "Analysis on cPCR results to see if we should send for sequencing ", "Colony PCR on CFP_SC1/SC2 (Max's transformants from Aug 25). Use primers 13&14 ", "Run products on a 1% gel to check if the PCR worked ", "Analysis on cPCR results to see if we should send for sequencing ", "Colony PCR on pSB1C3 with Gal1 and Adh1 (Cody's Transformants)", "Run products on a 1% gel to check if the PCR worked ", "Analysis on cPCR results to see if we should send for sequencing ", "Run PCR cleanup products from yesterday - CFP_NSC/SC1/SC2 and Gal1/Cup1 with 5&6 on a 1.2% gel", "Digest 1000ng CFP_NSC/SC1/SC2 with ApaI and BamHI for 35 mins ", "Column Purify CFP_NSC/SC1/SC2 and nanodrop", "Digest 2000ng X3 of Hsp104 plasmid with ApaI and BamHI for 2h at 37C", "Gel Extract 8.8kB fragment after running on a gel with guanosine. Other fragment should be 700bp", "Ligate Hsp104 with CFP_NSC/SC1/SC2. Use 100ng vector and 0.3uL ligase per reaction ", "Make 20 Amp plates (2 bottles of LB) ", "Restreak 4 transformants onto YPD plates and incubate at RT for the weekend ", "Take waste down to Greg "], "Aug27": ["Check Patch Plates from all of yesterday's cPCR", "Colony PCR on pXP218 Gal1. Use primers 15&16", "Run products on a 1% gel to check if the PCR worked ", "Analysis on cPCR results to see if we should send for sequencing ", "Clear out all the ice from the bottom of the freezer", "Wash dishes, clean lab benches and around the lab", "Colony PCR on CFP_NSC/SC1/SC2. Use primers 13&14"], "Aug24": ["Check colony PCR plates for red/white colonies and see if we have to inoculate any to send for sequencing ", "Inoculate colonies to send for sequencing & make a list with Alicia ", "Transformation of pSB1C3 with Gal1 and Adh1 into DH5alpha. New comp cells", "Pellet o/n CaCl2 cells, resuspend in 0.085M-15%glycerol", "Aliquot comp cells from yesterday ", "Transform 1, 2uL and 4uL of Strain111 (ie. pSB1C3-RFP) into the new comp cells. No +ve CTRL.", "PCR of CFP_NSC, CFP_SC1. 50uL reaction volumes. Do a -ve control ", "Run products on a 1.2% gel to see if PCR worked - run at 80-100V ", "PCR cleanup on the products, nanodrop ", "Digest 1000ng of CFP_NSC, CFP_SC1 with ApaI and BamHI for 35 min at 37C ", "Column purification of CFP_NSC, CFP_SC1 and nanodrop ", "Digest 2000ng of Hsp104 plasmid X2 with ApaI and BamHI for 2h at 37C ", "Gel Extract 8.8kB fragment after running on a gel with guanosine. Other fragment should be 700bp", "Ligate CFP_NSC, SC1 into Hsp104. Use 0.3uL ligase per rxn,100ng of the vector 3:1 insert:vector", "PCR of CFP_NSC, CFP_SC2. 50uL reaction volumes. Do a -ve control ", "Run products on a 1.2% gel to see if PCR worked - run at 80-100V ", "PCR cleanup on the products, nanodrop ", "Digest 1000ng of CFP_NSC, CFP_SC2 with ApaI and BamHI for 35 min at 37C ", "Column purification of CFP_NSC, CFP_SC2 and nanodrop ", "Digest 2000ng of Hsp104 plasmid X2 with ApaI and BamHI for 2h at 37C ", "Gel Extract 8.8kB fragment after running on a gel with guanosine. Other fragment should be 700bp", "Ligate CFP_NSC, SC2 into Hsp104. Use 0.3uL ligase per rxn,100ng of the vector 3:1 insert:vector", "PCR of Cup1 and Gal1 with primers 5&6. Use Patrick's parts and conditions (50uL volumes)", "Run products on a 1.2% gel to see if PCR worked - run at 80-100V ", "PCR cleanup of the products, nanodrop ", "Digest 2000ng of pXP218 with SphI and SalI X2. Digest for 2h at 37C ", "Gel extract 6.2kB fragment after running on a gel with guanosine. There should be only 1 band ", "Digest 1000ng of Cup1 and Gal1 (5&6 primers) with SphI and SalI for 35min at 37C", "Column purification of Cup1 and Gal1 then nanodrop ", "Ligate Cup1 and Gal1 into pXP218 and leave at RT for minimum 2h, 0.3uL ligase, 3:1 insert:vector ratio", "PCR of Gal1 and Adh1 with primers 17&18. Use synthesized parts and 50uL rxn volumes ", "Run products on a 1.2% gel to see if PCR worked - run at 80-100V ", "PCR cleanup of the products, nanodrop ", "Digest 2000ng of pSB1C3 with EcoRI and PstI, incubate at 37C for 1 hour ", "Gel extract 2kB fragment after running on a gel with guanosine. Other fragment will be 1kB ", "Digest Adh1 and Gal1 (17&18 primers) with EcoRI and PstI. Incubate at 37C for 35 min", "Column purification of Adh1 and Gal1 then nanodrop ", "Ligate Adh1 and Gal1 into pSB1C3, leave at RT minimum 2h, 0.3uL ligase/rxn. Do 2:1 ratio insert:vector ", "Digest 1000ng of CFP_SC1, CFP_SC2 with ApaI and BamHI for 35 min at 37C ", "Column purification of CFP_SC1, CFP_SC2 and nanodrop ", "Digest 2000ng of Hsp104 plasmid X2 with ApaI and BamHI for 2h at 37C ", "Gel Extract 8.8kB fragment after running on a gel with guanosine. Other fragment should be 700bp", "Ligate CFP_NSC, SC1 into Hsp104. Use 0.3uL ligase per rxn,100ng of the vector 3:1 insert:vector", "Digest HSP104 plasmid with BamHI and ApaI for 2hrs at 37 degrees", "Gel Extract 8.8kB fragment after running on a gel with guanosine. Other fragment should be 700bp", "Ligate HSP104 with CFP_NSC/SC1/SC2 and leave O/N at RT ", "Stuff tip boxes, autoclave, and return to storage.", "Clean office and throw out recycling/garbage.", "Wash glassware and wipe down bench tops. Clean up the lab (ie. sweep the floor)", "Autoclave new 1.5mL, 2mL, and PCR tubes, then dry oven."], "Aug25": ["Check if pSB1C3 transformation worked and decide on colony PCR ", "Colony PCR of the plates - 10 colonies per plate and use primers 19/20 ", "Run products on a 1% gel to check if we have the insert ", "Analysis to see if we should send for sequencing ", "Check efficiency test of the competent cells ", "Transformation of Hsp104_SC1/2 into DH5alpha competent cells ", "Transformation of Hsp104_NSC/SC1/2 into DH5alpha competent cells ", "Transformation of Hsp104_NSC/SC2 into DH5alpha competent cells ", "Transformation of Hsp104_NSC/SC1 into DH5alpha competent cells ", "Transformation of pXP218_Gal1/Cup1 into DH5alpha competent cells ", "Transformation of pSB1C3_Gal1/Adh1 into DH5alpha competent cells", "PCR of Cup1/Gal1 with Primers 5/6. Do 2X reactions ", "Run products on a 1% gel to check if PCR worked ", "PCR cleanup of the products, then nanodrop.", "PCR of CFP_NSC/SC1/SC2 with Primers 11/12. Do 2X reactions per synthesis ", "Run products on a 1% gel to check if PCR worked ", "PCR cleanup of the products, then nanodrop.", "Sequencing in! Do analysis to see if we have the products ", "Move WO plates from flowhood to our own storage space."], "Aug28": ["Colony PCR HSP NSC/SC1/SC2. Use primers 13&14", "Run products on a 1% gel to check if the PCR worked ", "Analysis on cPCR results to see if we should send for sequencing ", "Inoculate PSB ADH1 #3"], "Aug29": ["PCR of CFP_NSC,CFP_SC1, CFP_SC2. 50uL reaction volumes. Do a -ve control ", "Run products on a 1.2% gel to see if PCR worked - run at 80-100V ", "PCR cleanup on the products, nanodrop ", "Attempt 1: Digest 1000ng of CFP_NSC, CFP_SC1, CFP_SC2 with ApaI and BamHI for 45-60min at 37C ", "Attempt 1: Run a 1.4% gel on the digested products to see if they actually digested before CP", "Attempt 2: Digest 1000ng of CFP_NSC, CFP_SC1, CFP_SC2 with ApaI for 120min, then BamHI for 30min.", "Attempt 2: Run a 1.4% gel on the digested products to see if they actually digested before CP", "Column purification of CFP_NSC, CFP_SC1, CFP_SC2 and nanodrop ", "Attempt 1: Digest 2000ng of Hsp104 plasmid X3 with ApaI and BamHI for 120min at 37C ", "Attempt 2: Digest 2000ng of Hsp104 plasmid X3 with ApaI for 120min, then BamHI for 30min.", "Gel Extract 8.8kB fragment, then nanodrop. ", "Run a gel on the CPd/GEd insert/vector to double-check if we have what we want.", "Ligate CFP_NSC,SC1, SC2 into Hsp104.", "PCR of Cup1 and Gal1 with primers 5&6. Use Patrick's parts and conditions (50uL volumes)", "Run products on a 1.2% gel to see if PCR worked - run at 80-100V ", "PCR cleanup of the products, nanodrop ", "Digest 2000ng of pXP218 with SphI and SalI X2. Digest for 2h at 37C ", "Gel extract 6.2kB fragment after running on a gel with guanosine. There should be only 1 band ", "Digest 1000ng of Cup1 and Gal1 (5&6 primers) with SphI and SalI for 35min at 37C", "Column purification of Cup1 and Gal1 then nanodrop ", "Ligate Cup1 and Gal1 into pXP218 and leave at RT for minimum 2h, 0.3uL ligase, 3:1 insert:vector ratio", "PCR of Gal1 and Adh1 with primers 17&18. Use synthesized parts and 50uL rxn volumes ", "Run products on a 1.2% gel to see if PCR worked - run at 80-100V ", "PCR cleanup of the products, nanodrop ", "Digest 2000ng of pSB1C3 with EcoRI and PstI, incubate at 37C for 1 hour ", "Gel extract 2kB fragment after running on a gel with guanosine. Other fragment will be 1kB ", "Digest Adh1 and Gal1 (17&18 primers) with EcoRI and PstI. Incubate at 37C for 35 min", "Column purification of Adh1 and Gal1 then nanodrop ", "Ligate Adh1 and Gal1 into pSB1C3, leave at RT minimum 2h, 0.3uL ligase/rxn. Do 1:1 ratio insert:vector ", "Digest 1000ng of Cup1 and Gal1 (5&6 primers) with SphI and SalI for 35min at 37C", "Column purification of Cup1 and Gal1 then nanodrop ", "Ligate Cup1 and Gal1 into pXP218 and leave at RT for minimum 2h, 0.3uL ligase, 3:1 insert:vector ratio", "Make more working GelRed reagent from 5x stock.", "Attempt 1: PCR Amplify sgRNA-Main & RFP CTRL w/ 128 and 129, 50uL rxn volumes each.", "Make new working primers for 128 and 129, checking Benchling to confirm sequence.", "Attempt 2: PCR Amplify sgRNA-Main & RFP CTRL w/ 128 and 129, 50uL rxn volumes each.", "Inoculate 284 and 335 and in 5mL LB w/ Cm and Amp, respectively x5", "Make 10 Cm plates and 10 Cm/Amp plates reserved for BioBrick Improvement.", "Attempt 1: dGel of PCRd Main/RFP CTRL products in 1,4% gel.", "Attempt 2: dGel of PCRd Main/RFP CTRL products in 1,4% gel.", "Clean-up of the PCRd products, assuming they've worked, then nanodrop.", "Attempt 3: PCR Amplify sgRNA Main w/ new working stock, and RFP CTRL from synth. stock.", "Remove bioharzardous bench waste and clean counters with ethanol."], "Sep12": ["Run PCR NSC/SC1/SC2 on a gel (samples from last friday)", "PCR cleanup NSC/SC1/SC2", "Run cPCR SC1 from yesterday on a gel again just to get some nicer bands", "Transform Cup1 into HSP ", "Make 10 AMP plates", "Innoculate Psi- in YPD (x3)", "Innoculate Hsp NSC1 in Amp LB (x3)", "Streakplate Psi- on YPD plate", "Streakplate Hsp NSC1 on Amp LB plate"], "Sep13": ["Transform HSP NSC, SC1, SC2", "Miniprep pSB Gal1 2,5,6 and elute in H2O to send off for sequencing", "cPCR NSC/SC1/SC2-pSB Batch 4 (~8 colonies)", "Run products on a 1% gel to check if the PCR worked ", "cPCR Adh1-pXP Batch 4 (~8 colonies)", "Run products on a 1% gel to check if the PCR worked ", "Make 10 amp plates", "Sacrifice first year to the transformation gods (PRIORITY ALPHA)"], "Sep11": ["PCR amplify ADH1 and Gal1 using primers 25 and 26", "Digest 6000 ng pXP218 backbone with BamHI and SalI", "Run diagnostic gel of ADH1 and Gal1", "PCR clean up/column purify ADH1, Gal1, and pXP digest", "Digest ADH1 and Gal1 with BamHI and Gal1", "Column purify ADH1 and Gal1", "Ligate ADH1 and Gal1 into pXP backbone", "Ligate Cup1 into Hsp104 backbone (6:1 ratio)"], "Sep16": ["Run PCR products and digessted ADH1 and Gal1 from Sept 11 on a gel", "PCR amplify ADH1 and Gal1 with primers 25 and 26", "PCR clean up ADH1 and Gal1", "Digest ADH1 and Gal1", "Column purify ADH1 Gal1 digests", "PCR cup/gal with primers 5 and 6", "PCR SC2 with primers 11 and 12", "Run cup/gal/sc2 on gel to see if PCR worked", "PCR clean up cup and SC2"], "Sep17": ["Digest 1000ng of CFP SC2 with ApaI and BamHI for 1 hour ", "Column purification of CFP_SC1, CFP_SC2 and nanodrop ", "Digest 2000ng of Hsp104 plasmid X3 with ApaI and BamHI for 1 hour", "Gel Extract 8.8kB fragment, then nanodrop. ", "Ligate CFP_SC1, SC2 into Hsp104. 6:1 ratio, 0.3uL ligase per reaction", "Digest 1000ng of Cup1 (5&6 primers) with SphI and SalI for 1.5 hours at 37C", "Column purification of Cup then nanodrop ", "Digest 2000ng of pXP218 with SphI and SalI. Digest for 5h at 37C ", "Gel extract 6.2kB fragment after running on a gel with guanosine. There should be only 1 band ", "Ligate Cup1 and Gal1 into pXP218 and leave at RT for minimum 2h, 0.3uL ligase, 3:1 insert:vector ratio", "Miniprep Cup1-Hsp 3,17,21,25,29 and elute in H2O for sequencing", "Clean lab benchtops", "Wash dishes", "Digest ADH1 Gal1", "Digest ADH1 and Gal1", "Column purify ADH1 Gal1 digests", "Ligate ADH1 and Gal1 into pXP218 backbone"], "Sep14": ["Transform Sup35-GFP, HSP104-CFP NSC/SC1 into S. cerevisiae ", "Co-transform Sup35-GFP/HSP104 NSC and Sup35-GFP/HSP104 SC! into S. cerevisiae ", "Innoculate.....", "Miniprep 5mL of Hsp104-SC1 x2 and elute in elution buffer for transformation into yeast ", "Miniprep 3mL of Hsp104-SC1 x1 and elute in water for sequencing ", "Make frozen stock of Hsp104-SC1 and label appropriately in the strain list ", "Miniprep pSB-Gal1 #2,5,6", "Make frozen stocks of pSB-Gal1 #2,5,6 and label appropriately in strain list. Use 2mL tubes "], "Sep15": ["PCR amplify ADH1 and Gal1 using primers 25 and 26", "Run PCR products and digested ADH1 and Gal1 from Sept 11 on a gel", "PCR clean up ADH1 and Gal1", "Digest ADH1 and Gal1", "Column purify ADH1 Gal1 digests", "Make 2L LB broth", "Innoculate Cup_Hsp samples", "Make frozen stocks of pSB-Gal1 #2,5,6 and label appropriately in strain list. Use 2mL tubes ", "Innoculate Hsp104-SC1 x2 in Amp "], "Sep18": ["PCR Hsp NSC/SC1/SC2 using primers 21/22", "Run products on gel", "PCR cleanup products and nanodrop", "Plate yeast onto YPD", "Transform E. coli with pXP218_ADH1, pXP218_Gal1 + controls", "Make Amp plates", "Plate transformed E. coli on Amp", "PCR amplify Cup1, Gal1 and ADH1 with primers 25 & 26", "Run products on gel", "PCR cleanup products and nanodrop"], "Sep19": ["Image Gel of ADH1, Gal1, Cup1 PCR products from yesterday", "Colony PCR pXP218_ADH1 and pXP218_Gal1", "Inoculate all Yeast cultures for experiments tomorrow (Total 18 tubes) "], "Aug19": ["Ligate the backbone with CFP_Gal1, CFP-SC1 and CFP-SC2. Leave o/n at RT.", "Ligate pSB1C3 with the three inserts. Leave o/n at RT.", "PCR Gal1 and Cup1 using primers 5&6.", "Run the products on a 1.2% gel ", "PCR clean-up of the 2 inserts, then nanodrop.", "Re-run some CFP cPCR samples from yesterday on a 1% gel, check lab book to see which samples", "Make frozen stock with 1mL G9-inoculant using 40% Glycerol; label as Strain 393, and add to iGEM Box 6", "Miniprep inoculants 389-393 into sterile MilliQ, then nanodrop.", "Digest the miniprepped 393 w/ E and P (~250ng)", "Diagnostic gel on the digested 393t. Determine if we truely have the insert around 1.5kb", "Make a sequencing sheet for everyone to use and determine when to send stuff for sequencing.", "Prepare miniprepped samples for sending for sequencing ", "Establish a protocol for sgRNA-Main RFP experimetns with flow cytometer. "], "Aug18": ["Ligate the backbone with CFP_Gal1, CFP-SC1 and CFP-SC2. Leave o/n at RT.", "Ligate pSB1C3 with the three inserts. Leave o/n at RT.", "PCR Gal1 and Cup1 using primers 5&6.", "Run the products on a 1.2% gel ", "PCR clean-up of the 2 inserts, then nanodrop.", "Run ADH1 [Batch2] products from yesterday on a 1% gel", "Run pSB1C3 cPCR products of ADH1/Gal1 on a 1% gel.", "Make frozen stock with 1mL G9-inoculant using 40% Glycerol; label as Strain 393, and add to iGEM Box 6", "Inoculate Strain 389, 390, 391, 392 and 393 into 5mL LB + 2.5uL Cm each. Inc. o/n 37C.", "Miniprep 4mL G9-inoculant of pSB1C3-Gal [Batch2]", "Digest the miniprepped G9-inoculant of pSB1C3 w/ E and P (~250ng)", "Diagnostic gel on the digested G9-inoculant. Determine if we truely have the insert around 1.5kb", "Make a sequencing sheet for everyone to use and determine when to send stuff for sequencing.", "Establish a protocol for sgRNA-Main RFP experimetns with flow cytometer. "], "Aug17": ["Check if Transformation for Track A Batch 2/3 Worked - CFP NSC/SC1/SC2", "Check if Transformation for Track C pSB1C3 (Biobricks) worked ", "Check if Transformation for Track C pXP218 (Testing Plasmids) worked ", "Nanodrop the PCR products from the CFP reactions yesterday ", "Digest CFP_Gal1, CFP-SC1 and CFP-SC2 w/ ApaI and BamHI, using up to 2000ng of DNA if possible.", "Digest 4000ng of Hsp104 with BamHI and ApaI, use different reaction tubes.", "Column purify the three digested CFP fragments, then nanodrop.", "Gel extract the Hsp104 backbone, then nanodrop.", "Ligate the backbone with CFP_Gal1, CFP-SC1 and CFP-SC2. Leave o/n at RT.", "Ligate pSB1C3 with the three inserts. Leave o/n at RT.", "PCR Gal1, Cup1 using primers 5&6", "Miniprep G8 from the Inoculation yesterday then nanodrop ", "Digest 500ng of G8 with XbaI and PstI for a diagnostic digest - ", "Run products on a 1.2% gel to determine if the Gal1 insert is actually there! ", "Inoculate G9 from the Patch Plate of pSB1C3-Gal1 [Batch2] into LB w/ 2.5uL Cm", "Organize Freezer ", "cPCR on CFP-NSC, CFP-SC1 and CFP SC-2. There are 3 plates of each. Use primers 15&16", "cPCR on CFP-NSC, CFP-SC1 and CFP SC-2. There are 3 plates of each. Use primers 15&16", "cPCR on pSB1C3-ADH1/Gal1 w/ primers 19/20", "cPCR on pXP218-Cup1/Gal1 w/ primers 14/15", "Inoculate G9 from the Patch Plate of pSB1C3-Gal1 [Batch2] into LB w/ 2.5uL Cm", "Run products on a 1% gel at 110V for 45min-1hours on the cPCRs", "Analyze gel to determine if any of these experiments worked! ", "Make a sequencing sheet for everyone to use and determine when to send stuff for sequencing.", "Establish a protocol for sgRNA-Main RFP experimetns with flow cytometer. "], "Aug16": ["PCR amplify CFP-NSC, CFP-SC1, and CFP-SC2.", "Diagnostic 1.2% gel of the amplified products. ", "PCR clean-up of the three CFP inserts, then nanodrop.", "Transform Track A ligation products (NSC/SC1/SC2) from Aug15 onto Amp plates.", "Transform Track C pSB1C3 ligation products (ADH1/Cup1) from Aug15 onto Cm plates.", "Transform Track C pXP218 ligation products (Gal1/Cup1) from Aug15 onto Amp plates.", "Digest CFP_Gal1, CFP-SC1 and CFP-SC2 w/ ApaI and BamHI, using up to 4000ng of DNA if possible.", "Digest 6000ng of Hsp104 with BamHI and ApaI, use different reaction tubes.", "Column purify the three digested CFP fragments, then nanodrop.", "Gel extract the Hsp104 backbone, then nanodrop.", "Ligate the backbone with CFP_Gal1, CFP-SC1 and CFP-SC2. Leave o/n at RT.", "Digest pSB1C3, ADH1, Gal1 and Cup1 w/ EcoRI and PstI, up to 2000ng if possible. ", "Column purify the three digested inserts, then nanodrop.", "Gel extract the 2kb fragment of pSB1C3, then nanodrop.", "Ligate pSB1C3 with the three inserts. Leave o/n at RT.", "Digest pXP218, Gal1, and Cup1 w/ SalI and SphI, up to 2000ng if possible. ", "Column purify the two inserts, then nanodrop.", "Gel extract the larger fragment of pXP218 once ran on a 1.2% gel, then nanodrop.", "Ligate pXP218 with the two inserts. Leave o/n at RT.", "Inoculate G8 from the Patch Plate of pSB1C3-Gal1 [Batch2] into LB w/ 2.5uL Cm", "Organize freezer", "Establish a protocol for Sup35:GFP measurements for gene retention with flow cytometry.", "Establish a protocol for sgRNA-Main RFP experimetns with flow cytometer. "], "Aug15": ["URGENT: Check the transformants from Aug12 transformation of TrackA Batch1/2 of the CFP-Gal1/SC1/SC2", "URGENT: Check the transformants from Aug13 transformation of TrackC Batch1 of the Gal1/Cup1 in pXP218", "PCR amplify ADH1, Gal1, and Cup1 with 17 and 18.", "Make new working stock of primers 17&18", "PCR amplify ADH1, Gal1, and Cup1 with 5 and 6.", "Run the products on a 1.2% gel ", "PCR clean-up of the six inserts, then nanodrop.", "Analyze transformants of pSB1C3-Gal1/-Cup1 and determine if cPCR is appropriate.", "Analyze transformants of CFP-Gal1/-SC1/-SC2 and determine if cPCR is appropriate.", "Digest CFP_Gal1, CFP-SC1 and CFP-SC2 w/ ApaI and BamHI, using up to 4000ng of DNA if possible.", "Digest 6000ng of Hsp104 with BamHI and ApaI, use different reaction tubes.", "Column purify the three digested CFP fragments, then nanodrop.", "Gel extract the Hsp104 backbone, then nanodrop.", "Ligate the backbone with CFP_Gal1, CFP-SC1 and CFP-SC2. Leave o/n at RT.", "Digest pSB1C3, ADH1, and Cup1 w/ EcoRI and PstI, up to 2000ng if possible. ", "Column purify the three digested inserts, then nanodrop.", "Gel extract the 2kb fragment of pSB1C3, then nanodrop.", "Ligate pSB1C3 with the three inserts. Leave o/n at RT.", "Digest pXP218, Gal1, and Cup1 w/ SalI and SphI, up to 2000ng if possible. ", "Column purify the two inserts, then nanodrop.", "Gel extract the larger fragment of pXP218 once ran on a 1.2% gel, then nanodrop.", "Ligate pXP218 with the two inserts. Leave o/n at RT.", "Colony PCR on Transformation plate of pSB1C3_ADH1 fragment. Pick 10+ colonies. Use primers 19&20.", "Run a gel 1% on all 10 products and controls (controls are empty plasmid and no DNA)", "Analyze gel and Determine if ADH1-GFP got into pSB1C3 backbone", "Colony PCR on Transformation plate of pSB1C3_Gal1 fragment. Pick 10+ colonies. Use primers 19&20.", "Run a gel 1% on all 10 products and controls (controls are empty plasmid and no DNA)", "Analyze gel and Determine if GAL1-GFP got into pSB1C3 backbone", "Wash glassware", "Fill 10uL tip boxes (x12) only to discover the autoclave was shut down for the week.", "Establish a protocol for Sup35:GFP measurements for gene retention with flow cytometry.", "Establish a protocol for sgRNA-Main RFP experimetns with flow cytometer. "], "Aug14": ["Make Amp plates if required for transformation", "Make Cm plates if required for transformation", "Transform Hsp104_NoStop, SC1 and SC2 as well as controles into E. coli, plate on Amp", "Transform pSB1C3_ADH1 and pSB1C3_Gal1 as well as controls into E. coli, plate on Cm", "Colony PCR on Transformation plate of pSB1C3_ADH1 fragment. Pick 10+ colonies. Use primers 19&20.", "Run a gel 1% on all 10 products and controls (controls are empty plasmid and no DNA)", "Analyze gel and Determine if ADH1-GFP got into pSB1C3 backbone", "Colony PCR on Transformation plate of pSB1C3_Gal1 fragment. Pick 10+ colonies. Use primers 19&20.", "Run a gel 1% on all 10 products and controls (controls are empty plasmid and no DNA)", "Analyze gel and Determine if GAL1-GFP got into pSB1C3 backbone", "Wash glassware", "Establish a protocol for Sup35:GFP measurements for gene retention with flow cytometry.", "Establish a protocol for sgRNA-Main RFP experimetns with flow cytometer. "], "Aug13": ["PCR amplify CFP-Gal1, CFP-SC1, and CFP-SC2.", "Run the products on a 1.2% gel ", "PCR clean-up of the three CFP inserts, then nanodrop.", "Digest CFP_Gal1, CFP-SC1 and CFP-SC2 w/ ApaI and BamHI, using up to 4000ng of DNA if possible.", "Column purify the three digested CFP fragments, then nanodrop.", "Digest 6000ng of Hsp104 with BamHI and ApaI, use different reaction tubes.", "Gel extract the Hsp104 backbone, then nanodrop.", "Ligate the backbone with CFP_Gal1, CFP-SC1 and CFP-SC2. Leave o/n at RT.", "Digest pSB1C3, ADH1 andGal1 w/ EcoRI and PstI, up to 2000ng if possible. ", "Column purify the two digested inserts, then nanodrop.", "Gel extract the 2kb fragment of pSB1C3, then nanodrop.", "Ligate pSB1C3 with the three inserts. Leave o/n at RT.", "Make working primer tubes for primers 19&20, see primer list (these are from 2014)", "Colony PCR on Transformation plate of pSB1C3_ADH1 fragment. Pick 10+ colonies. Use primers 19&20.", "Run a gel 1% on all 10 products and controls (controls are empty plasmid and no DNA)", "Analyze gel and Determine if ADH1-GFP got into pSB1C3 backbone", "Colony PCR on Transformation plate of pSB1C3_Gal1 fragment. Pick 10+ colonies. Use primers 19&20.", "Run a gel 1% on all 10 products and controls (controls are empty plasmid and no DNA)", "Analyze gel and Determine if GAL1-GFP got into pSB1C3 backbone", "Transform the ligation products of pXP218 and Cup1/ Gal1.", "Wash glassware", "Establish a protocol for Sup35:GFP measurements for gene retention with flow cytometry.", "Establish a protocol for sgRNA-Main RFP experimetns with flow cytometer. "], "Aug12": ["PCR amplify CFP-Gal1, CFP-SC1, and CFP-SC2.", "Run the products on a 1.2% gel ", "PCR clean-up of the three CFP inserts, then nanodrop.", "Transform the ligation products of CFP-Gal1/-SC1/-SC2.", "Digest CFP_Gal1, CFP-SC1 and CFP-SC2 w/ ApaI and BamHI, using up to 4000ng of DNA if possible.", "Column purify the three digested CFP fragments, then nanodrop.", "Digest 6000ng of Hsp104 with BamHI and ApaI, use different reaction tubes.", "Gel extract the Hsp104 backbone, then nanodrop.", "Ligate the backbone with CFP_Gal1, CFP-SC1 and CFP-SC2. Leave o/n at RT.", "Make 20 Amp plates (200uL Amp/ LB-Agar bottle).", "Run 1% diagnostic on pSB1C3-Gal1 digested product.", "PCR amplify Patrick's Gal1 and Cup1 using exact same recipe & conditions as ADH1.", "Run a 1.2% diagnostic gel on the PCR products above. 80V, 45mins is enough,", "PCR Clean-up of the Gal1/Cup1 amplification products according to gel results, then nanodrop.", "Digest pSB1C3, ADH1 andGal1 w/ EcoRI and PstI, up to 2000ng if possible. ", "Column purify the three digested inserts, then nanodrop.", "Gel extract the 2kb fragment of pSB1C3, then nanodrop.", "Ligate pSB1C3 with the three inserts. Leave o/n at RT.", "Digest pXP218 with SalI and SphI, up to 2000ng if possible. ", "Digest Cup1 and Gal1 w/SalI and SphI, 1500ng ", "Column purify Gal1, then nanodrop.", "Gel extract the larger fragment of pXP218 once ran on a 1.2% gel, then nanodrop.", "Ligate pXP218 with the two inserts. Leave o/n at RT.", "Colony PCR on Transformation plate of ADH1 fragment. Pick 10 colonies. Use primers 15&16.", "Run a gel 1.2% 80V on all 10 products and control ", "Analyze gel and Determine if ADH1-GFP got into pXP backbone", "Make frozen stock of the four pSB1C3-Cup1 cultures in the 37C incubator. ", "Miniprep approximately 3mL from each of those cultures into MilliQ, then nanodrop.", "Fill 200ul tip boxes", "Make working primer tubes for primers 19&20, see primer list (these are from 2014)", "Colony PCR on Transformation plate of pSB1C3_ADH1 fragment. Pick 10+ colonies. Use primers 19&20.", "Run a gel 1% on all 10 products and controls (controls are empty plasmid and no DNA)", "Analyze gel and Determine if ADH1-GFP got into pSB1C3 backbone", "Colony PCR on Transformation plate of pSB1C3_Gal1 fragment. Pick 10+ colonies. Use primers 19&20.", "Run a gel 1% on all 10 products and controls (controls are empty plasmid and no DNA)", "Analyze gel and Determine if GAL1-GFP got into pSB1C3 backbone", "Establish a protocol for Sup35:GFP measurements for gene retention with flow cytometry.", "Establish a protocol for sgRNA-Main RFP experimetns with flow cytometer. "], "Aug11": ["PCR Clean-up of Peter's amplified CFP-SC1 and CFP-SC2, then nanodrop.", "Digest CFP_Gal1, CFP-SC1 and CFP-SC2 w/ ApaI and BamHI, using up to 4000ng of DNA if possible.", "Column purify the three digested CFP fragments, then nanodrop.", "Digest 6000ng of Hsp104 with BamHI and ApaI, use different reaction tubes.", "Gel extract the Hsp104 backbone, then nanodrop.", "Ligate the backbone with CFP_Gal1, CFP-SC1 and CFP-SC2. Leave o/n at RT.", "Digest 200ng of pSB-Gal1 using EcoRI and PstI", "Run diagnostic digest of pSB-Gal1 on 1% agarose gel, 1ul sample, 4 water, 2 dye. Use gel red.", "PCR Clean-up of Patrick's Gal1 and Cup1, then nanodrop.", "PCR amplify Patrick's Gal1 and Cup1 using exact same recipe & conditions as ADH1.", "Run a 1.2% diagnostic gel on the PCR products above. 80V, 45mins is enough,", "PCR Clean-up of the Gal1/Cup1 amplification products according to gel results, then nanodrop.", "Digest pSB1C3, ADH1, Gal1, and Cup1 w/ EcoRI and PstI, up to 2000ng if possible. ", "Column purify the three digested inserts, then nanodrop.", "Gel extract the 2kb fragment of pSB1C3, then nanodrop.", "Ligate pSB1C3 with the three inserts. Leave o/n at RT.", "Digest pXP218, Gal1, and Cup1 w/ SalI and SphI, up to 2000ng if possible. ", "Column purify the two inserts, then nanodrop.", "Gel extract the larger fragment of pXP218 once ran on a 1.2% gel, then nanodrop.", "Ligate pXP218 with the two inserts. Leave o/n at RT.", "Colony PCR on Transformation plate of ADH1 fragment. Pick 10 colonies. Use primers 15&16.", "Run a gel 1.2% 80V on all 10 products and control ", "Analyze gel and Determine if ADH1-GFP got into pXP backbone", "Organize freezer/ working boxes. ", "Design new primers for amplification of ADH1, CUP1, and GAL1 into pXP218 "], "Aug10": ["Resuspend CFP primers and make working primer stocks", "PCR CFP-Gal1, and CFP stopcodons 1 and 2", "Run PCR products on a 1.2% gel and run at 80V ", "Digest 1000 ng of each CFP, 4000 ng Hsp104 with ApaI and BamHI", "Column purify CFP digest, gel extract Hsp digest", "Ligate Hsp104 and CFP, CFP-stop1 and CFP-stop2", "Organize freezer", "PCR amplify Cup1 & Gal1 using primers 5&6", "Diagnostic 1.4% agarose on PCR priducts of Cup1/Gal1", "Design new primers for amplification of ADH1, CUP1, and GAL1 into pXP218 ", "Digest 200ng of pSB-Gal1 using EcoRI and PstI", "Run diagnostic digest of pSB-Gal1 on 1% agarose gel, 1ul sample, 4 water, 2 dye. Use gel red", "PCR amplfy Cup1 using primers 17&18", "Run PCR products on a 1% gel at 80V", "PCR amplify gal1 and cup1 from previous step using primers 5&6", "Run PCR products on a 1% gel at 80V", "Clean lab benches and fill microtubule containers, make and autoclave 40% glycerol solution"], "Sep25": ["cPCR pXP Gal1 and pXP ADH1", "Run products on gel"], "Sep24": ["Transform Promoter Characterization Constructs from yesterday into E. coli", "Make Amp plates", "Plate Promoter Characterization Transformants"], "Sep23": ["PCR Amplify ADH1 (Primers 25 &26), Gal1, Cup1 (Primers 5&6)", "PCR clean up ADH1, Gal1, Cup1", "Digest ADH1, Gal1, Cup1, pXP218", "Column purify ADH1, Gal1, Cup1, pXP218 digests", "Ligate ADH1, Gal1, Cup1, into pXP218"], "Sep22": ["PCR amplify Gal1 with primers 25 & 26, from Gal1 stock using TaqMM", "Make 2L LB-Agar, put in 200 ml bottles and autoclave", "Run PCR products on gel", "Clean PCR products"], "Sep20": ["PCR amplify ADH1 with primers 25 & 26, Gal1 and Cup1 with primers 5 & 6", "Run PCR products on gel", "Clean PCR products"], "Jul1": ["4 Pellet using 3K RPM, 10min, 4C; decant; resuspend in 4mL 0.085M CaCl2 - 15% Glycerol", "5 Make 100uL aliquots into ~40 1.5mL tubes, and refirgerate at -80C", "Make 1L YPD and autoclave in flasks to prepare for transformation ", "Clean test tubes, already soaking in sparkcleen in white bucket, scrub and rinse with DI, & autoclave", "Troubleshoot the oePCR on SnapGene", "Test competency of the new comp cells with transformation of pSB1C3-RFP plasmids."], "Jul7": ["PCR amplify the ADH1:GFP fragment using Primers 5 & 6", "Diagnostic gel on the PCR product, 1.4% agarose gel run at 80V ", "Use PCR Clean up kit for the PCR product +Nanodrop", "Transform the ligation products of CFP_Gal1 w/ Hsp104", "Figure out activation parameters for the Gal1 promoter (get help from squad C)", "Design SDM primers for removing SpeI site from Hsp104 ORF & plan experiment ", "Design synthesized part with premature stop codon ", "Streak plate yeast transformation onto YPD (2 colonies per plate) then grow ", "Write an experimental plan for going from PSI- to PSI+ and how often we will take samples ", "Fill p10 tip boxes (one full bag), autoclave, and place in the dry oven o/n,"], "Jul6": ["PCR amplify the ADH1:GFP fragment using Primers 5 & 6", "Diagnostic gel on the PCR product, 1.4% agarose gel run at 80V ", "Use PCR Clean up kit for the PCR product +Nanodrop", "Clean labware in sink"], "Jul5": ["PCR amplify the ADH1:GFP fragment using Primers 5 & 6", "Diagnostic gel on the PCR product, 1% agarose gel run at 80V ", "Use PCR Clean up kit for the PCR product +Nanodrop", "Restore lab benches to their original conditions (clean them the hell up).", "Make 1L 50x TAE and then refill the 1X TAE jug. "], "Jul4": ["Return autoclaved material and reagents back into storage and shelves. ", "PCR amplify the ADH1:GFP fragment using Primers 5 & 6", "Diagnostic gel on the PCR product, 1% agarose gel run at 80V ", "Use PCR Clean up kit for the PCR product +Nanodrop", "Order new primers for the oePCR group", "PCR of CFP_Gal1 with primers 7&8 (3 tubes, 0.5uL DNA in each, Tm 70C, Extension time 45s) ", "Diagnostic gel on the PCR product (should be 1.4 kB long), 0.8% agarose gel ", "PCR Cleanup of CFP_Gal1 (3X tubes) fragment and nanodrop ", "Digest 1000ng of Hsp104 and CFP_Gal1 with XhoI and BamHI", "Run a diagnostic gel on Hsp104 plasmid to see bands if digest worked ", "Gel Extract Hsp104 fragment from the digest ", "Column purification of both fragments and nanodrop ", "Ligate CFP_Gal1 into Hsp104 plasmid using 3:1 ratio ", "Design primers for biobricking Hsp104-CFP into pSB1C3", "Inoculate Hsp104 leu plasmid for miniprep tomorrow 5X tubes with Amp ", "Inoculate sup35 plasmid for miniprep tomorrow 5X tubes with Amp ", "Replica plate PSI+ strain again - should be ade2-, leu2-, his3-, trp3-, ura3- ", "Restreak PSI- strain on YPD media (RT)", "Inoculate PSI- yeast strain in 5mL YPD x5 for transformation tomorrow (in 30 C)", "Define activation parameters for the Cup1 promoter to induce the response ", "Make 20 Amp agar plates"], "Jul9": ["Miniprep the inoculants from yesterday (x9)", "Autoclave the test tubes and place in dry oven o/n."], "Jul8": ["Do analysis on transformation yesterday - think about next steps ", "Inoculate 9 transformants on appropriate antibiotics and incubate o/n at 37C", "PCR amplify Gal1_CFP from the stock. Use 0.5uL of DNA template, 70C annealing, 45s elongation", "Run the Gal1_CFP on a 1% agarose gel at 80V. 1uL product, 4uL water, 1uL LD", "PCR Cleanup of the Gal1_CFP fragment and store in the -20C freezer", "Make streak plates (single colonies) from the patches of the transformation plates from yesterday", "Design synthesized part with premature stop codon ", "Design experiment of PSI- to PSI+ and Cup1 parameters to test out our system", "PCR amplify the ADH1:GFP fragment using Primers 5 & 6", "Diagnostic gel on the PCR product, 1% agarose gel run at 80V ", "Use PCR Clean up kit for the PCR product +Nanodrop", "Remove tip boxes from the dry oven", "Wash labware"], "Sep8": ["PCR of CFP SC1/SC2 with primers 11 and 12 ", "Run products on a 1% gel to see if the PCR worked ", "PCR cleanup of HSP SC1/SC2 and nanodrop ", "Digest 1000ng of CFP SC1/SC2 with ApaI and BamHI for 1 hour ", "Column purification of CFP_SC1, CFP_SC2 and nanodrop ", "Digest 2000ng of Hsp104 plasmid X3 with ApaI for 2 hours, then BamHI for 30min.", "Gel Extract 8.8kB fragment, then nanodrop. ", "Ligate CFP_SC1, SC2 into Hsp104. 6:1 ratio, 0.3uL ligase per reaction", "PCR of CFP NSC/SC1/SC2 with primers 21 and 22 ", "Run products on a 1% gel to see if the PCR worked ", "PCR cleanup of CFP NSC/SC1/SC2 and nanodrop ", "Digest 1000ng of CFP SC1/SC2 with EcoRI and PstI for 1 hour at 37C", "Column purification of CFP_NSC, CFP_SC1, CFP_SC2 and nanodrop ", "Digest 2000ng of pSB1C3 with EcoRI and PstI at 37C for 1 hour ", "Gel extract 2kB fragment, then nanodrop ", "Ligate CFP NSC/SC1/SC2 into pSB1C3 using a 1:1 ratio, 0.3uL ligase per reaction", "Prepare pSB1C3-Gal1 samples for sequencing tomorrow ", "Inoculate the 5 Hsp104-NSC that worked for miniprep and sequencing tomorrow", "Inoculate cultures for transformation tomorrow into yeast ", "PCR of Cup1 primers 23 and 24", "Run products on a 1% gel to see if the PCR worked ", "PCR cleanup of Cup1 and nanodrop ", "Digest 1000ng of Cup1 with ApaI and SacI for 1 hour at 37C", "Column purification of Cup1 and nanodrop ", "Digest 2000ng of Hsp104 with ApaI and SacI for at 37C for 1 hour ", "Gel extract 2kB fragment, then nanodrop ", "Ligate Cup1 with Hsp104 using a 5:1 ratio, 0.3uL ligase per reaction", "PCR of Cup1 primers 5 and 6", "Run products on a 1% gel to see if the PCR worked ", "PCR cleanup of Cup1 and nanodrop "], "Sep9": ["Miniprep inoculated samples ", "Frozen stocks ", "Prepare Gal sampels to sequence ", "Prepare Hsp104-NSC samples to sequence ", "Prepare sgRNa samples to sequence", "Send order for sequencing ", "PCR of CFP_NSC/SC1/SC2 with primers 21 and 22 ", "Digest 1000ng of Cup1 with ApaI and SacI for 1 hour at 37C", "Column purification of Cup1 and nanodrop ", "Digest 2000ng of Hsp104 with ApaI and SacI for at 37C for 1 hour ", "Gel extract 6kB fragment, then nanodrop "], "Sep1": ["cPCR CFP NSC/SC1/SC2-Hsp104 Batch 2 (~8 colonies)", "Run products on a 1% gel to check if the PCR worked ", "cPCR Cup1/Gal1-pXP Batch 3 (~8 colonies)", "Run products on a 1% gel to check if the PCR worked ", "cPCR ADH1/Gal1-pSB Batch 2 (~8 colonies)", "Run products on a 1% gel to check if the PCR worked ", "cPCR sgRNA-Main & RFP-CTRL (~50 colonies)", "Run products on a 1% gel to check if the PCR worked ", "Attempt 2: cPCR CFP NSC/SC1/SC2-Hsp104 Batch 1: A2/B2/C2 Set & A1(1-4,6)", "Run products on a 1% gel to check if the PCR worked, ONLY LOAD 1UL OF SAMPLE", "Fill p200 tips!!! We're out now!!! Autoclave!!!", "Make 1L of LB Agar (in 5-200mL bottles), and autoclave.", "PCR of CFP_NSC,CFP_SC1, CFP_SC2. 50uL reaction volumes. Do a -ve control ", "Run products on a 1.2% gel to see if PCR worked - run at 80-100V ", "PCR cleanup on the products, nanodrop ", "Digest 1000ng of CFP_NSC, CFP_SC1, CFP_SC2 with ApaI for 240min, then BamHI for 30min.", "Run a 1.4% gel on the digested products to see if they actually digested before CP", "Column purification of CFP_NSC, CFP_SC1, CFP_SC2 and nanodrop ", "Digest 2000ng of Hsp104 plasmid X3 with ApaI for 240min, then BamHI for 30min.", "Gel Extract 8.8kB fragment, then nanodrop. ", "Ligate CFP_NSC,SC1, SC2 into Hsp104.", "PCR of Cup1 and Gal1 with primers 5&6. Use Patrick's parts and conditions (25 rxn volumes)", "Run products on a 1.2% gel to see if PCR worked - run at 80-100V ", "PCR cleanup of the products, nanodrop ", "Digest 2000ng of pXP218 with SphI and SalI X2. Digest for 2h at 37C ", "Gel extract 6.2kB fragment after running on a gel with guanosine. There should be only 1 band ", "Digest 1000ng of Cup1 and Gal1 (5&6 primers) with SphI and SalI for 35min at 37C", "Column purification of Cup1 and Gal1 then nanodrop ", "Ligate Cup1 and Gal1 into pXP218 and leave at RT for minimum 2h, 0.3uL ligase, 3:1 insert:vector ratio", "PCR of Gal1 and Adh1 with primers 5&6. Use synthesized parts and 25 rxn volumes ", "Run products on a 1.2% gel to see if PCR worked - run at 80-100V ", "PCR cleanup of the products, nanodrop ", "Digest 2000ng of pSB1C3 with EcoRI and PstI, incubate at 37C for 1 hour ", "Gel extract 2kB fragment after running on a gel with guanosine. Other fragment will be 1kB ", "Digest Adh1 and Gal1 (17&18 primers) with EcoRI and PstI. Incubate at 37C for 35 min", "Column purification of Adh1 and Gal1 then nanodrop ", "Ligate Adh1 and Gal1 into pSB1C3, leave at RT minimum 2h, 0.3uL ligase/rxn. Do 1:1 ratio insert:vector "], "Sep2": ["cPCR CFP NSC/SC1/SC2-Hsp104 Batch 2 (~8 colonies)", "Run products on a 1% gel to check if the PCR worked ", "cPCR Cup1/Gal1-pXP Batch 3 (~8 colonies)", "Run products on a 1% gel to check if the PCR worked ", "Transform the Cup1/Gal1-pXP Batch 4", "Transform the ADH1/Gal1-pSB Batch 3", "Make 2L of LB Agar (in 5-200mL bottles), and autoclave.", "Inoculate M1-1,3,8;M2-1,2,4,6,8;M3-4,5,8 into 5mL LB w/ 2.5uL Cm", "Digest 1000ng of CFP_NSC, CFP_SC1, CFP_SC2 with ApaI for 240min, then BamHI for 30min.", "Run a 1.4% gel on the digested products to see if they actually digested before CP", "Column purification of CFP_NSC, CFP_SC1, CFP_SC2 and nanodrop ", "Digest 2000ng of Hsp104 plasmid X3 with ApaI for 240min, then BamHI for 30min.", "Gel Extract 8.8kB fragment, then nanodrop. ", "Ligate CFP_NSC,SC1, SC2 into Hsp104.", "Digest 2000ng of pSB1C3 X2 with EcoRI and PstI, incubate at 37C for 1 hour ", "Gel extract 2kB fragment after running on a gel with guanosine. Other fragment will be 1kB ", "Digest Adh1 and Gal1 (17&18 primers) with EcoRI and PstI. Incubate at 37C for 35 min", "Column purification of Adh1 and Gal1 then nanodrop ", "Ligate Adh1 and Gal1 into pSB1C3, leave at RT minimum 2h, 0.3uL ligase/rxn. Do 1:1 ratio insert:vector ", "PCR of Gal1 with primers 5&6. Use Patrick's parts and conditions (25 rxn volumes)", "Digest 2000ng of pXP218 with SphI and SalI X2. Digest for 2h at 37C ", "Gel extract 6.2kB fragment after running on a gel with guanosine. There should be only 1 band ", "Digest 1000ng of Cup1 and Gal1 (5&6 primers) with SphI and SalI for 35min at 37C", "Column purification of Cup1 and Gal1 then nanodrop ", "Ligate Cup1 and Gal1 into pXP218 and leave at RT for minimum 2h, 0.3uL ligase, 3:1 insert:vector ratio", "Eat 120 chicken nuggets (PRIORITY ALPHA)", "Pour agar plates with CM antibiotic", "Make more 1x TAE buffer for gels"], "Sep3": ["Transform ADH1/Gal-pSB Batch 4", "Transform the Cup1/Gal1-pXP Batch 5", "Miniprep Pat's shit", "cPCR Cup1/Gal1-pXP Batch 4 (~8 colonies)", "Run products on a 1% gel to check if the PCR worked ", "cPCR Adh1/Gal1-pSB Batch 3 (~8 colonies)", "Run products on a 1% gel to check if the PCR worked ", "PCR of ADHI with primers 25&26. Use Patrick's parts and conditions (25 rxn volumes)", "Run products on a 1.2% gel to see if PCR worked - run at 80-100V ", "PCR cleanup of the products, nanodrop ", "Digest 2000ng of pXP218 with BamHI and SalI. Digest for 2h at 37C ", "Gel extract 6.2kB fragment after running on a gel with guanosine. There should be only 1 band ", "Digest 1000ng of ADHI (25&26 primers) with BamHI and SalI for 35min at 37C", "Column purification of ADHI then nanodrop ", "Ligate Cup1 and Gal1 into pXP218 and leave at RT for minimum 2h, 0.3uL ligase, 3:1 insert:vector ratio", "PCR of ADHI with primers 25&26. Use Patrick's parts and conditions (25 rxn volumes)", "Run products on a 1.2% gel to see if PCR worked - run at 80-100V ", "PCR cleanup of the products, nanodrop ", "Let dirty dishes soak overnight in soap and water", "Clean dirty counters", "Clean gel microwave"], "Sep4": ["cPCR Adh1/Gal1-pSB Batch 3 (~8 colonies)", "Run products on a 1% gel to check if the PCR worked ", "cPCR Cup1/Gal1-pXP Batch 4 (~8 colonies)", "Run products on a 1% gel to check if the PCR worked ", "Digest 1000ng of ADHI (25&26 primers) with BamHI and SalI for 35min at 37C", "Column purification of ADHI then nanodrop ", "Ligate ADH1 into pXP218 and leave at RT for minimum 2h, 0.3uL ligase, 3:1 insert:vector ratio", "Digest 1000ng of CFP_NSC, CFP_SC1, CFP_SC2 with ApaI for 240min, then BamHI for 30min.", "Run a 1.4% gel on the digested products to see if they actually digested before CP", "Column purification of CFP_NSC, CFP_SC1, CFP_SC2 and nanodrop ", "Digest 2000ng of Hsp104 plasmid X3 with ApaI for 2 hours, then BamHI for 30min.", "Gel Extract 8.8kB fragment, then nanodrop. ", "Digest 3000ng of Hsp104 plasmid X3 with ApaI for 1 hour, then BamHI for 30min.", "Gel Extract 8.8kB fragment, then nanodrop. ", "Ligate CFP_NSC,SC1, SC2 into Hsp104.", "Transfer soapy dishes into diH2O and let them soak"], "Sep5": ["Transform HSP NSC, SC1, SC2", "Transform pXP Adh1", "Digest 1000ng of CFP_NSC, CFP_SC1, CFP_SC2 with ApaI for 240min, then BamHI for 30min.", "Column purification of CFP_NSC, CFP_SC1, CFP_SC2 and nanodrop ", "Digest 2000ng of Hsp104 plasmid X3 with ApaI for 2 hours, then BamHI for 30min.", "Gel Extract 8.8kB fragment, then nanodrop. ", "Ligate CFP_NSC,SC1, SC2 into Hsp104.", "Inoculate pSB Gal1 2,5,6"], "Sep7": ["cPCR NSC/SC1/SC2-pSB Batch 5 (~8 colonies)", "Run products on a 1% gel to check if the PCR worked ", "Prepare sgRNA-Main sequencing protocol."], "Jun17": ["Make 100mL 0 5M EDTA pH 8 0 autoclave", "Make 50mL TE Buffer 10mM Tris HCl pH 8 + 1mM EDTA pH 8 then filter sterilize", "Rearrange & order 4 remaining IDT sequences ", "Organize SnapGene files ", "Get alphaW303 yeast strain to use at PSI control and streak on plate ", "Streak Duncker lab strains on YPD ", "Streak frozen stocks to test", "Make new Tris HCl 100ml, 1 0M and pH of 8 0"], "Jun16": ["Make frozen stocks"], "Jun19": ["Innoculate cultures for longterm storage stock plasmids Hsp104/Sup35/pXP ", "Innoculate culture for Hsp104 b/Leu plasmid miniprep"], "Jun30": ["Check Transformation plates from yesterday's transformation and do analysis ", "Replica plate PSI+ and PSI- strains to make sure they have correct genotype", "Make 1L YPD and autoclave in flasks to prepare for transformation ", "Refill blue tip box with 10ul tips and autoclave for Dr. Moffatt", "Clean test tubes, already soaking in sparkcleen in white bucket, scrub and rinse with DI, & autoclave", "Label the damn drawers already cause I have no clue where glassware is now"]}